Simultaneous analysis of a large number of biological samples is useful in various types of analysis, for example, flow cytometry, DNA sequencing, liquid chromatography, oligonucleotide analysis, and various electrophoretic techniques. Rapid DNA analysis is of particular importance in the Human Genome Project, which is an attempt to identify the sequence of bases in human DNA.
One technique that has been applied to the sequencing of DNA is capillary electrophoresis. In this technique, an appropriate solution is polymerized or gelled to form a porous matrix in a fused silica capillary tube of internal dimensions in the order of 50 .mu.m. An electric field is then applied across the matrix. Fragments of sampled DNA injected into one end of the capillary tube migrate through the matrix under the effect of the electric field at speeds that depend on the length of the fragment. Therefore, different length fragments arrive at a detection part of the capillary at different times. The dideoxynucleotide at one end of the fragment may be labelled with a fluorescent marker during a reaction step. The fluorescent marker is associated with the terminating dideoxynucleotide. When the fragment passes through a beam of light from a laser in a detection zone, the fluorescent marker fluoresces and the fluorescence may be detected as an electric signal. The intensity of the electric signal depends on the amount of fluorescent marker present in the matrix in the detection zone. The dideoxynucleotide at the end of the fragment may then be identified by a variety of methods. As different length fragments migrate through the matrix under the applied field, a profile of the fragments may be obtained.
A multiple capillary biochemical analyzer for use in capillary electrophoresis and for other applications is disclosed in our U.S. Pat. No. 5,439,578 issued Aug. 8, 1995. In that patent a multiple capillary analyzer is disclosed which, among its other features, discloses detection of light from multiple capillaries which terminate in a flow chamber. Sheath fluid entrains individual sample streams from the capillaries, and collimated sample excitation radiation is applied simultaneously across the ends of the capillaries. Light emitted from the excited sample is detected by an optical detection system. The disclosure and drawings of said patent are hereby incorporated in their entirety by reference into this specification.
In one embodiment of the analyzer disclosed in the above-identified patent, the rows of capillaries are offset, with the furthest back row of capillaries furthest downstream, so that the rows of capillaries in effect form a staircase. This offset configuration allows samples migrating from a number of rows of multiple capillaries to be imaged simultaneously, without overlap, onto photo detectors. Imaging occurs through one of the walls of the cuvette.
There are several disadvantages to the staircase configuration disclosed. First, the rows of capillaries in the back of the cuvette are imaged through a millimeter or more of sheath fluid, while the capillaries in the front of the cuvette are imaged through only a few micrometers of fluid. The resultant difference in optical path lengths leads to optical aberration. While the aberration can be largely corrected by including a prism in the optical train, it cannot easily be entirely corrected.
Secondly, stray laser light illuminates the capillaries, leading to background light scatter and fluorescence. While careful adjustment of the illumination conditions can be used to try to correct this problem, a two-dimensional array of capillaries is inherently more sensitive to light scatter than a single dimensional array of capillaries. However a two-dimensional array is preferred so that samples from a larger number of capillaries can simultaneously be analyzed.
Thirdly, it is desirable for the capillaries to be uniformly spaced, to obtain good sheath flow and uniformly spaced sample streams, and so that the position of each fluorescence spot will be known and will not overlap a non-fluorescing spot. Achievement of this uniform spacing is extremely difficult to obtain.
Accordingly, it is an object of the invention in one of its aspects to produce a multiple capillary analyzer which can alleviate some of the above disadvantages. To this end the invention provides in one of its aspects an analyzer for analyzing an organic sample, said analyzer comprising:
(a) a plurality of capillary tubes arranged side by side, each capillary tube having first and second ends, the second ends of the capillary tubes terminating adjacent each other and the first ends being connectable to a source of organic sample, PA1 (b) a flow chamber having an interior cavity, the second ends of the capillary tubes terminating inside the interior cavity, PA1 (c) means to force said organic sample through the capillary tubes from the first ends of the capillary tubes to the second ends of the capillary tubes, PA1 (d) means to provide sheath fluid into the interior cavity of said flow chamber to provide a flow of sheath fluid past the second ends of the capillary tubes and for entraining organic sample from said capillary tubes in individual sample streams from the second ends of the capillary tubes, PA1 (e) a barrier member spaced from the second ends of said capillary tubes, said barrier member including a plurality of openings therein, said openings being aligned with said second ends of said capillary tubes for the individual sample streams therefrom to pass through said openings, said barrier member having a first side facing said second ends of said capillary tubes, and a second side opposite said first side, PA1 (f) radiation means providing electromagnetic radiation having a wavelength that may excite said sample to emit radiation, said radiation means being positioned to illuminate said sample streams between said second ends of said capillary tubes and said first side of said barrier member, PA1 (g) and radiation detection means on said second side of said barrier means for detecting radiation which is emitted from said sample streams and which passes through said openings to said second side of said barrier member.
Further objects and advantages of the invention will appear from the following description, taken together with the accompanying drawings.